mTOR Regulates Endocytosis and Nutrient Transport
in Proximal Tubular Cells
Florian Grahammer,* Suresh K. Ramakrishnan,†Markus M. Rinschen,‡Alexey A. Larionov,† Maryam Syed,†Hazim Khatib,§Malte Roerden,§Jörn Oliver Sass,|¶Martin Helmstaedter,* Dorothea Osenberg,* Lucas Kühne,* Oliver Kretz,* Nicola Wanner,* Francois Jouret,** Thomas Benzing,‡Ferruh Artunc,§Tobias B. Huber,*††‡‡and Franziska Theilig†
*Department of Medicine IV, Medical Center—University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany;†Institute of Anatomy, Department of Medicine, University of Fribourg, Fribourg, Switzerland;
‡Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne,
Germany;§Department of Medical IV, Sektion Nieren- und Hochdruckkrankheiten, University of Tübingen, Tübingen,
Germany;|Bioanalytics and Biochemistry, Department of Natural Sciences, Bonn Rhein Sieg University of Applied Sciences, Rheinbach, Germany;¶Division of Clinical Chemistry and Biochemistry and Children’s Research Centre,
University Children’s Hospital Zürich, Zurich, Switzerland; **Groupe Interdisciplinaire de Génoprotéomique Appliquée, Cardiovascular Sciences, University of Liège, Liege, Belgium; and††BIOSS, Centre for Biological Signalling Studies and
‡‡FRIAS, Freiburg Institute for Advanced Studies and ZBSA, Center for Biological System Analysis, Albert Ludwigs
University of Freiburg, Freiburg, Germany
ABSTRACT
Renal proximal tubular cells constantly recycle nutrients to ensure minimal loss of vital substrates into the urine. Although most of the transport mechanisms have been discovered at the molecular level, little is known about the factors regulating these processes. Here, we show that mTORC1 and mTORC2 specifically and synergistically regulate PTC endocytosis and transport processes. Using a conditional mouse genetic ap-proach to disable nonredundant subunits of mTORC1, mTORC2, or both, we showed that mice lacking mTORC1 or mTORC1/mTORC2 but not mTORC2 alone develop a Fanconi-like syndrome of glucosuria, phosphaturia, aminoaciduria, low molecular weight proteinuria, and albuminuria. Interestingly, proteomics and phosphoproteomics of freshly isolated kidney cortex identified either reduced expression or loss of phosphorylation at critical residues of different classes of specific transport proteins. Functionally, this resul-ted in reduced nutrient transport and a profound perturbation of the endocytic machinery, despite preserved absolute expression of the main scavenger receptors, MEGALIN and CUBILIN. Ourfindings highlight a novel mTOR–dependent regulatory network for nutrient transport in renal proximal tubular cells.
With the transition from water to land, mammalian kidneys developed into a high–pressure filtration system combining excess ultrafiltration with highly efficient tubular reabsorption to minimize nutrient and protein loss.1The vast amount of the ultra fil-trate (approximately 60%–70%), including virtu-ally all nutrients, is already being reabsorbed by the first nephron segment, the proximal tubule (PT).2 Specific loss of PT amino acid or phosphate trans-port function is associated with genetically defined disease entities, such as Hartnup disorder or hypo-phosphatemic rickets.3,4In contrast, disrupted en-ergy metabolism or toxic injury can simultaneously
affect several transport systems, leading to a Fanconi syndrome.5 , 6 In addition to aminoaciduria,
T.B.H. and F.T. contributed equally to this work.
Correspondence: Dr. Franziska Theilig, Department of Medi-cine, University of Fribourg, Route Albert-Gockel 1, 1700 Fri-bourg, Switzerland or Dr. Tobias B. Huber, Medical Center, University of Freiburg, Center of Medicine, Renal Division, Breisacherstrasse 66, 79106 Freiburg, Germany. Email: franziska. theilig@unifr.ch or tobias.huber@uniklinik-freiburg.de
1VCMJTIFEJO+PVSOBMPGUIF"NFSJDBO4PDJFUZPG/FQISPMPHZ
EPJ"4/
glucosuria, and phosphaturia, Fanconi syndrome is charac-terized by low molecular weight proteinuria.7This is caused by dysfunction of receptor-mediated endocytosis, which is mediated by the two scavenger receptors, MEGALIN and CUBILIN, followed by clathrin-mediated internalization of the ligand-receptor complex.8Fusion with early endosomes delivers the complex to the endosomal-lysosomal com-partment, leading to recycling or degradation of retrieved proteins.8 The prominent endocytotic activity of the PT is important to retrieve vital plasma proteins and maintain body homeostasis.9Although individual components of this system have been recently described, little is known on the organ-specific regulation of these transport processes.
The mammalian homolog of TOR1 (mTORC1) and mTORC2 are intracellular multiprotein complexes regulating tissue–specific cellular functions.10The regulatory associated protein of mammalian target of rapamycin (RAPTOR) is im-portant to assemble mammalian target of rapamycin (mTOR) complex 1, and its function can be blocked by rapamycin. In contrast, mTORC2, containing the rapamycin-insensitive companion of mammalian target of rapamycin (RICTOR) as a scaffolding protein, seems to be insensitive to short– term rapamycin treatment.11mTORC1 integrates a wide va-riety of signals, including growth factors, amino acids, cellular energy content, and cellular stress, to regulate cellular growth and division as well as lipid and mitochondrial biogenesis.12,13 The wide range of mTORC1 phosphorylation targets is still incompletely defined but seems to vary in a cell-specific man-ner. Conversely, mTORC2 seems to be activated by insulin and related pathways and controls several downstream AGC ki-nases by phosphorylating their hydrophobic motif.14,15 Thereby, mTORC2 could play an important role in cell sur-vival and organization of the cytoskeleton.
To investigate the effect of mTOR-controlled signaling in PT function, we generated inducible conditional Raptor, Rictor, or double-knockout mice in renal tubular cells along the nephron.
RESULTS
Loss of mTOR in PT Cells Results in a Fanconi-Like Phenotype
Expression of the mTORC1 and mTORC2 complexes in the PT was verified by staining for mTOR, RAPTOR, S6K1, ribosomal S6 protein (S6P), and N-Myc Downstream Regulated 1 (NDRG1) (Supplemental Figure 1). All components were strongly expressed in the S1 and S2 segments of the PT in either overlay or close vicinity to MEGALIN. The proximal tubular S3 segment only showed minor signal intensity for mTOR, S6K1, and S6P and was devoid of signals for RAPTOR and NDRG1. To examine the effects of mTORC deletion on PT function, we generated mice with doxycycline–inducible Pax8 promotor– driven tubular deletion of Raptor (Raptorfl/fl 3 Pax8rtTA 3 TetOCre; subsequently termed RapDTubule), Rictor (Rictorfl/fl 3 Pax8rtTA 3 TetOCre; subsequently termed RicDTubule), or both
genes (Raptorfl/fl 3 Rictorfl/fl 3 Pax8rtTA 3 TetOCre; subse-quently termed RapRicDTubule) (Figure 1, A–C). Protein abun-dance or reduced phosphorylation of known downstream targets was used to assess efficiency of our knockout approach (Figure 1, D–F). In comparison with the respective control group, all exam-ined parameters were at least reduced by 70% (RapDTubule: RAPTOR,274.6%64.6%; p-S6P/S6P, 278.9%66.4%; P,0.05; n=6; RicDTubule: RICTOR, 284.5%68.4%; p-NDRG1/ NDRG1,271.1%63.7%; P,0.05; n=6; RapRicDTubule: RAPTOR, 278.3%66.2%; RICTOR, 269.1%65.3%; p-S6P/S6P, 289.2% 64.7%; p-NDRG1/NDRG1, 271.7%69.8%; P,0.05; n=6) (Fig-ure 1, D–F).
Measurement of urinary glucose and phosphate excretion in RapRicDTubulemice revealed 3.3- andfivefold increases in urinary losses of glucose and phosphate, respectively, in-dicating impaired nutrient reabsorption (Figure 1H). In addition, SDS-PAGE of urinary samples revealed a slight increase in albuminuria in RapDTubule, which was significant in RapRicDTubule(Figure 1, I and K). This observation was confirmed by direct measurement of mouse albumin in uri-nary samples (Figure 1L). Furthermore, an increase in low molecular mass (LMW) proteinuria between 25 and 68 kD could be detected in RapRicDTubule(+126%637%; P,0.05; n=5) as well as a significant increase in proteinuria at ap-proximately 25 kD in RapDTubuleand RapRicDTubuleanimals (RapDTubule: +70%613%; RapRicDTubule: +44%66%; P,0.05; n=5). In addition, a pronounced increase in urinary amino acid excretion was observed in RapDTubuleanimals, and an even more pronounced increase was in RapRicDTubule an-imals but not in RicDTubule mice (Figure 1M, Supplemental Figure 2A). Amino acid plasma concentrations were similar in all experimental groups (Supplemental Figure 2B), confirming a renal amino acid transport defect rather than defects in hepatic or muscular metabolism.
Compared with control mice, RapDTubuleand RapRicDTubule animals showed significantly higher food and fluid intakes, which were associated with increased urinary volume and sodium excretion (Supplemental Table 1). No alterations were found in RicDTubule animals. Together, these data show a significant mTORC1–dependent defect in tubular reabsorption of glucose, phosphate, amino acids, LMW pro-teins, and albumin.
mTOR Regulates PTC Endocytosis and Intracellular Trafficking In Vivo
To gain additional insights into the effects of gene knockout on PT endocytosis, mice were injected with horseradish perox-idase (taken up by fluid-phase endocytosis) and Alexa555-coupled lactoglobulin (reabsorbed by receptor-mediated endocytosis) before perfusionfixation.16Morphologic anal-ysis revealed a strong reduction offluid-phase endocytosis in RapDTubuleand RapRicDTubuleanimals, whereas no change was observed in RicDTubulemice (Figure 2A). Similarly, confocal microscopy analysis showed a reduced receptor–mediated en-docytosis in RapDTubuleand RapRicDTubulemice in comparison
Figure 1. Proximal tubular deletion of mTORC1 leads to a Fanconi like syndrome. (A–C) Schematic of recombination strategy and site of inducible Pax8rtTA3 TetOCre–mediated (A) Raptor, (B) Rictor, and (C) double knockout within the tubular system. (D–F) Dem-onstration of knockout efficacy by Western blot of (D) RAPTOR and downstream target p-S6P/S6P in RapDTubule, (E) RICTOR and the
downstream target p-NDRG1/NDRG1 in RicDTubule, and (F) RAPTOR and RICTOR and downstream targets p-S6P/S6P and p-NDRG1/ NDRG1 in RapRicDTubule. (G) Urinary glucose (gluc) and phosphate (PO432) excretions in RapRicDTubule were increased. (H and I)
Coomassie staining of SDS-PAGE prepared from urine samples loaded after normalization to urinary creatinine concentration from (H) RapDTubuleand (I) RapRicDTubule. (J) Urinary albumin excretion of RapDTubuleand RapRicDTubule. (K) Urinary neutral amino acid excretion of RapDTubule, RicDTubule, and RapRicDTubule. ACR, albumin-creatinine-ratio; Con, control; crea, creatinine. *P value versus control ,0.05; **P value versus control ,0.01.
with respective control animals (Figure 2B). Introducing an mT/ mG reporter allele into RapDTubule animals revealed very few events of a mosaic Cre expression in PT cells. However, these few mosaic areas allowed a detailed cell to cell analysis of endo-cytosis. Cre–positive, Raptor–deficient cells in comparison with Cre–negative, Raptor–positive cells showed a highly impaired receptor–mediated endocytosis as identified by reduced uptake of Alexa555 lactoglobulin (Figure 2C). To quantify receptor– mediated endocytotic flux, fluorimetric measurements of endocytosed Alexa555 lactoglobulin in tissue homogenates were performed. Reduced endocytoticfluxes were observed in RapDTubuleand RapRicDTubuleanimals, with no difference in RicDTubulemice (Figure 2D). To examine intracellular traffick-ing, we stained for cellular endogenous albumin distribution.
RapDTubulemice and even more RapRicDTubulemice showed an accumulation of endogenous albumin with increases in ves-icle size and number (Figure 2E). This is in agreement with reduced intracellular trafficking and protein processing. Be-cause MEGALIN and CUBILIN are the main receptors for PT endocytosis, their expression pattern and abundance were analyzed. Immunoblotting and staining analyses revealed no change between groups in either subcellular localization or expression level (Supplemental Figure 3).
Taken together, these data show that mTOR function is required for PT endocytosis and intracellular trafficking, whereas the abundance of the apical scavenger receptors seemed unaffected. To analyze whether duration of kinase deletion could play a role, we assessed histologic alterations in Figure 2. Deletion of mTORC1 leads to a reducedfluid-phase as well as receptor-mediated endocytosis and blocks intracellular trafficking. (A) Fluid-phase and (B) receptor-mediated endocytosis is shown by the uptake of horseradish peroxidase or Alexa555-coupled lactoglobulin (red stain), respectively, injected 5 minutes before perfusion-fixation. Reduced uptake is found in RapDTubuleand
RapRicDTubule. (C) Cell by cell analysis of receptor-mediated endocytosis in RapDTubule3 mT/mG mice. Cre-positive cells (green stain) show reduced endocytic uptake of Alexa555-coupled lactoglobulin (red vesicular staining) in comparison with Cre-deficient cells. (D) Quantification of Alexa555-coupled lactoglobulin of RapDTubule, RicDTubule, and RapRicDTubulenormalized to the respective control mice.
Significantly reduced uptake is shown for RapDTubuleand RapRicDTubuleanimals. (E) Assessment of endogenous albumin content in
RapDTubule, RicDTubule, and RapRicDTubule. Note the increased number and size of albumin-containing vesicles. In immunofluorescence images, kidney sections were counterstained with Alexa647 phalloidin (blue stain) to mark actinfilaments of the BBM. Con, control. Scale bar, 20mm. **P,0.01.
RapDTubuleand RapRicDTubulemice 3 months after induction. In RapDTubuleand RapRicDTubule(much stronger) animals, the renal cortex was reduced, which was mainly caused by shrink-age of the convoluted part of the PT (Supplemental Figure 4A). MEGALIN and CUBILIN expression levels remained un-altered in RapDTubule but were decreased in RapRicDTubule (Supplemental Figure 4, B and C). We next analyzed the ex-pression pattern of the Na+/K+-ATPase (NKA), a basolateral protein being expressed in a polarized fashion. Interestingly, in RapDTubuleand RapRicDTubuleanimals 3 months postinduc-tion, apical NKA–containing vesicles were observed, showing an apical mis-sorting of this protein. In addition, we discerned a striking reduction of basolateral NKA expression in RapRicDTubuleanimals. These data show that apical scaven-ger receptor expression remained intact in RapDTubulemice, although signs of epithelial depolarization could be detected, whereas PT cells dedifferentiated in RapRicDTubule animals, leading to reduced MEGALIN and CUBILIN expression. Loss of mTOR Results in Altered Apical Plasma Membrane Topology with a Reduced Reabsorption SurfaceIn Vivo
Light and electron microscopy analyses were performed to correlate reduced PT function with changes in morphology. Periodic acid–Schiff staining on paraffin sections revealed re-duced brush border membrane (BBM) microvilli in all trans-genic knockout mice, which was most evident in RapDTubule and RapRicDTubulecompared with control animals (Figure 3A). The overall gross morphology, however, remained un-changed as shown in methylene blue–stained semithin sec-tions (Figure 3B). Subcellular electron microscopy analysis of PT S1 segments in RapDTubuleand RapRicDTubulemice showed reduced microvilli length and endosomal vesicle formation compared with control animals as well as reduced numbers of mitochondria in RapRicDTubuleanimals (Figure 3C, Supple-mental Table 2). Additionally, largely inflated early and late endosomes and Golgi cisternae were observed in RapDTubule and RapRicDTubulemice.
Reduced Endocytosis Is Caused by Diminished Formation of Clathrin-Coated Vesicles
To gain additional mechanistic insights, we analyzed endocy-tosis in the well established opossum kidney proximal tu-bule cell (OKC) line.17OKCs were incubated with 100 nM rapamycin (mTORC1 inhibitor), 100 nM PP242 (mTORC1+2 inhibitor), or 250 nM torin (mTORC1+2 inhibitor) for 3 days or 3 or 10mM PF-4708671 (S6K1 inhibitor) overnight before endocytosis assays followed byflow cytometric analysis of en-docytosed Alexa647 albumin. Similar to our in vivofindings, treatment with rapamycin, PF-4708671, PP242, or torin led to a strong reduction of endocytosed Alexa647 albumin (Sup-plemental Figure 5A). Increasing concentrations of rapamycin reduced the endocytosis rate in a concentration-dependent manner (Supplemental Figure 5B). To allow specific differen-tiation between mTORC1 and mTORC2 signaling, we
performed shRNA–mediated viral knockdown of the main mTORC1–dependent translational regulator S6K1, RICTOR, or both genes together. Compared with mock transduction, all virally transduced OKCs showed a significant reduction in their endocytosis rate, with the strongest decrease observed in S6K1 or S6K1/RICTOR shRNA knockdown (Supplemental Figure 5, C and D). mTORC1 and mTORC2 signaling showed no influence on lysosomal pH and enzyme activity (Supple-mental Figure 5E). In addition, enzymatic activity of galac-tosidase, mannosidase, and a-glucuronidase remained unchanged on viral knockdown of S6K1, RICTOR, or S6K1/ RICTOR compared with mock transduction (data not shown). Formation of clathrin-coated vesicles as a readout of membrane formation was reduced in S6K1 and double shRNA–transduced OKCs (Supplemental Figure 5, F and G). This may affect the formation of endocytotic vesicles as de-scribed in the electron microscopic analysis of the transgenic mouse models.
Proteomic and Phosphoproteomic Analyses of Kidney Cortex Identify a Putative mTOR–Dependent
Transport Regulatory Protein Network
To further elucidate the molecular causes for the observed reduction in PT transport function, we performed detailed proteomic and phosphoproteomic analyses of renal cortex in RapDTubuleand respective control animals. Although overall differences between control and RapDTubule animals were rather subtle (Figure 4A), several amino acid transporters, such as SLC6A19 (B0AT1), SLC7A7 (y+LAT1), and SLC3A2 (4F2hc), as well as proteins related to polarity and endocytosis (e.g., DOCK8, Bcl2-like 1 [BCL-xL], Ras–related protein Rab-10 [RAB10], and sorting nexin 8 [SNX8]) were reduced (Figure 4B, Supplemental Table 3). Making use of the mosaic expression pattern of RAPTOR-deficient cells in RapDTubule, we could confirm by immunohistochemistry the reduced ex-pression of B0AT1, y+LAT1, 4F2hc, DOCK8, and BCL-xLin RAPTOR-deficient cells in comparison with wild-type cells (Figure 5, A–E). This was corroborated by Western blot anal-ysis from isolated cortical kidney membranes (Figure 5F), which confirmed our proteomic findings.
Interestingly, BBM-bound enzymes, such as 59 nucleotid-ase, glutamylaminopeptidnucleotid-ase, and angiotensin-converting enzyme 2, or mitochondrial proteins, such as cytochrome P450 4A12A and cytochrome b-c1 complex subunit 8, were upregulated, likely in a compensatory fashion in RapDTubulemice (Figure 4B). We carried out gene ontology enrichment of the changed protein population compared with the unchanged protein population. Several nutrient transport pathways in the PT were again found to be significantly downregulated in RapDTubulemice (Figure 4C). Smallfiltered proteins taken up by the PT (e.g., transthyretin, angiotensinogen, retinol binding protein, vitamin D binding protein, andb2-microglobulin) were enriched in the renal cortexes of RapDTubuleanimals com-pared with controls (Figure 4D, Supplemental Table 3). Over-all analysis of phosphosites again only showed subtle
differences between the two genotypes (Figure 4E). However, detailed analysis revealed reduced phosphorylation of several transport proteins, especially amino acid transporter SLC7A9 (b0,+AT) and the sodium–dependent glucose transporter 2 (SGLT2; SLC5A2) providing additional potential molecular ex-planation for the observed aminoaciduria and glucosuria (Fig-ure 4F, Supplemental Table 4). Additionally, an up to eight times reduction on three so far unknown phosphosites of the Na+/HCO32cotransporter (NBC1; T3, S232, and S1060) and the ArfGEF GBF-1 (S179) were identified. To further evaluate the functional importance of the later protein, we used the
Drosophila melanogaster system to perform an siRNA knock-down in nephrocytes, the highly endocytotic storage kidney cells of thefly. Interestingly, knockdown of GBF-1 led to a marked reduction in endogenous uptake of a fluorescently labeled marker protein (Figure 5G). In line, TEM showed a phenotype consistent with a severely deranged endocytotic machinery (Figure 5H).
In contrast, several mTORC2 targets, especially NDRG1, were found to have an increased phosphorylation status (T328 and S342). Similar to the gene ontology enrichment analysis on protein level, transmembrane transport activity as well as Figure 3. mTOR deletion leads to ultrastructural alterations in PT cells. (A) Periodic acid–Schiff–stained sections of control (Con), RapDTubule,
RicDTubule, and RapRicDTubuleshowing reduced BBM length in mutants compared with control mice. (B) Semithin sections counterstained with methylene blue from Con, RapDTubule, RicDTubule, and RapRicDTubuleshowing intact morphology of epithelial cells. (C) Electron mi-croscopy images of Con, RapDTubule, RicDTubule, and RapRicDTubuleshowing reduced number and length of BBM microvilli and reduced number of clathrin-coated vesicles as well as recycling endosomes in otherwise normal cellular structures in mutant compared with control animals. Note the increase in early and late endosomes in mutant cells compared with Con in C. Scale bar, 20mm and 2 mm.
membrane components were most significantly regulated (Figure 4G). Interestingly, when integrating both proteomic and phosphoproteomic datasets, it became evident that changes in phosphorylation were not a mere consequence of changes in protein abundance (Figure 4H). Wefinally analyzed phosphomotifs being up- or downregulated. Downregulated phosphorylation sites mainly contained a proline-directed mo-tif ([S/T]–P), a momo-tif consistent with phosphorylation by mTOR but also, other proline–directed kinases (Figure 4I).18 Phosphorylation sites containing the AGC Kinase motif RxxS were increased.
DISCUSSION
Although the pleiotropic functions of mTOR kinases have recently become evident, very little is known about their complex role in regulating epithelial protein transport and endocytosis. Interestingly, registry datasets after renal trans-plantation document reduced serum potassium and phos-phorus levels in sirolimus-treated patients,19–21 pointing toward an mTOR regulatory function for electrolyte transport and homeostasis. Using inducible, gene–targeted, specific dis-ruption of mTORC1, mTORC2, or both mTOR complexes in Figure 4. Proteomic analyses of RapDTubulemice shows reduced abundance and phosphorylation of both tubular transport proteins and regulators of endocytosis. (A) Hierarchical clustering of identified proteins and the samples on the basis of the protein LFQ intensities. (B) Volcano plot of the proteomic analysis of RapDTubulemice compared with the control mice. Proteins beyond the curved lines were considered to be significantly changed (red, increased; blue, decreased). (C) Analysis of over–represented gene ontology (GO) terms in the decreased and increased protein populations compared with the unchanged protein population (Fisher exact test; size of the squares represents number of condensed terms). (D) Cumulative histogram of all quantified proteins (black) and proteins with the uniprot keyword secreted (red). Subgroups of this protein population with small molecular weight are depicted in magenta. (E) Hi-erarchical clustering of identified phosphorylation site intensity. (F) Volcano plot of the intensity of phosphorylation sites in the RapDTubulemice compared with the control mice. Phosphorylation sites beyond the curved lines were considered to be significantly changed. (G) Analysis of over–represented GO terms in the significantly changed phosphorylation site population compared with the unchanged population (Fisher exact test). (H) Comparison of logarithmized expression ratios (RapDTubuleto control) for proteins and their respective phosphorylation sites. Density is color coded. (I) Position-weighted matrix of phosphorylation motifs present in the increased and decreased phosphorylation site populations. GOCC, go-term cellular component; GOMF: go-term molecular function; LFQ, label free quantification; TTA: trans-tubular-absorption.
Figure 5. Reduced expression of transport proteins and regulators of endocytosis can be found in proximal tubular cells of RapDTubule mice. For better illustration, RapDTubule3 mT/mG mice were used for analysis of expression levels of various proteins by cell to cell analysis of Cre-positive (green stain) compared with wild-type cells. (A) B0AT1, (B) y+Lat1, (D) DOCK8, and (E) BCL-x
Lshowed strongly
reduced expression levels in Cre-positive cells compared with wild-type cells. Borders between cells types are marked with white bars. (C) In the case of 4F2hc, Alexa555 lactoglobulin was used to distinguish between Cre-positive cells and wild-type cells, and borders are marked by white bars. Cre-positive cells with reduced uptake of Alexa555 lactoglobulin also showed reduced basolateral ex-pression level of 4F2hc. (F) Western blots of B0AT1, y+Lat1, 4F2hc, DOCK8, and BCL-x
Lfrom control (Con) and RapDTubule(left panel)
and densitometric evaluation (right panel) confirm immunohistochemical results. (G) RNAi-mediated knockdown of Gbf-1 (CG8487; gartenzwerg) was generated in Drosophila, and RFP-ANP (red)fluorescence uptake was measured in nephrocytes (green). Quantifi-cation is presented in right panel. (H and I) Transmission electron microscopy images of Drosophila from (H) control and (I) Gbf-1 knockdown are shown. In comparison with control nephrocytes with a proper formation of the endocytotic machinery, Gbf-1 knockdown shows loss of clathrin vesicles and early, late, and recycling endolysomes, whereas the formation of aberrant vesicular structures is increased. Scale bar, 20mm in A–E; 2 mm in H; 0.2 mm in H, inset; 0.5 mm in I; 0.25 mm in I, inset. *P value versus control ,0.05; **P value versus control ,0.01. RFP-ANP, red-fluorescent-protein atrial natriuretic peptide; RNAi, RNA interference.
PT cells, we can now delineate the influence of mTOR signal-ing on PT physiology, protein transport, and endocytosis with unprecedented detail (Figure 6). In fact, disruption of mTORC1 signaling leads to a Fanconi-like syndrome, which presents with phosphaturia, glucosuria, aminoaciduria, and LMW proteinuria. On a molecular level, both fluid-phase and receptor–mediated endocytosis are impaired and coincide with an altered BBM length and vesicle formation in the PT. mTOR Regulates the Endocytosis Machinery in PT Epithelial Cells
Tubular albuminuria and LMW proteinuria are well estab-lished features of Fanconi syndrome and known to occur in patients treated with mTOR inhibitors after renal transplan-tation.22,23 Our study now highlights a significantly reduced tubular internalization in RapDTubuleand RapRicDTubulemice, which was consistent with endocytosis assays performed in vitro. Surprisingly, MEGALIN and CUBILIN, which are the main renal scavenger receptors forfiltered urinary proteins, were not directly regulated by mTOR in our functional studies (short–term follow-up after induced mTOR deletion).9This is in contrast to recently published data, in which long–term (6 months) rapamycin treatment was shown to lead to a reduced MEGALIN expression in mice.24However, it has been recog-nized that long-term administration of rapamycin in high doses additionally inhibits mTORC2 and exerts cytotoxic ef-fects.25In agreement, long–term follow-up studies (3 months) in our double–knockout RapRicDTubulemice showed a reduced MEGALIN expression, whereas RapDTubulemice maintained a stable MEGALIN expression. Additionally, we detected a strong reduction in the overall renal cortex volume because of shortening of the S1/2 segment in RapDTubulemice, and it was more pronounced in RapRicDTubulemice, confirming the importance of mTOR signaling for general cellular programs, such as cell growth, proliferation, and metabolism.
In addition to the impairment of the initiating steps of endocytosis, we also observed a disturbed endocytic trafficking as indicated by increased amounts of endocytosed albumin in RapDTubuleand RapRicDTubuleanimals. Furthermore, proteo-mic analysis of RapDTubule identified increased intracel-lular amounts of LMW proteins, such as angiotensinogen, retinol binding protein, vitamin D binding protein, and b2-microglobulin. These proteins are physiologically endocy-tosed by PT cells and further processed for degradation or transcytosis.26 Intracellular trafficking is a prerequisite for both processes, and its disturbances may, hence, lead to in-tracellular accumulations as observed in our animal models (Figure 2).
Proteomic data of RapDTubulekidney cortex did identify re-duced expression or phosphorylation level of interesting can-didate proteins, such as BCL-xL, GBF-1, SNX8, RAB10, and DOCK8, to explain the reduced endocytosis rate, altered api-cal membrane differentiation, and impaired intracellular traf-ficking. BCL-xLis an antiapoptotic protein with a role in the regulation of membrane dynamics and endocytosis.27
DOCK8, a member of the evolutionarily conserved DOCK family proteins,28 was shown to bind to nucleotide-free CDC42 to mediate the GTP-GDP exchange reaction. Thereby, CDC42 is spatially activated at the plasma membrane,29where it plays a crucial role in apical differentiation and BBM for-mation.30 Another GEF, the ArfGEF GBF-1, presented a strongly diminished phosphorylation status in RapDTubule animals. Arf-GEFs are nucleotide exchange factors for Arf GTPases, which have diverse functions in vesicle coat forma-tion and vesicle-cytoskeleton interacforma-tions.31SNX8 was found to localize at early endosomes, and its knockdown was shown to affect intracellular vesicle trafficking.32 RAB10 has been localized to many organelles, such as endoplasmic reticulum (ER), Golgi, early endosomes, and recycling endosomes, and mediates membrane trafficking within the cell partly by reg-ulating endosomal phosphatidylinositol-4,5-bisphosphate levels.33,34Together, these data reveal a novel theme of mTOR function by regulating a series of proteins being specifically in-volved in endocytosis and vesicle transport.
mTOR Regulates Specific Apical Tubular Transport Proteins
Many BBM transporters were affected by Raptor deletion, re-sulting in a Fanconi-like syndrome. SGLT2 (SLC5A2) is the main apical glucose transporter in the PT S1 and S2 segments, with an exclusive localization to the BBM.35The transporter activity is decisively regulated by phosphorylation.36 Phos-phoproteomics of RapDTubulemice pointed to a reduced phos-phorylation of SGLT2 at S623 in comparison with control animals. This residue is a highly conserved phosphosite between species, including human SGLT2 (S624).36 Aug-mented phosphorylation at this site was shown to enhance glucose transport by increasing membrane insertion of SGLT2.36 Loss of mTORC1-induced phosphorylation at S623 could, hence, contribute to glucosuria in RapDTubule and RapRicDTubuleanimals.
RapDTubuleand even more so, RapRicDTubule mice dis-played a highly increased excretion of neutral, basic, and acidic amino acids compared with control mice (Figure 1, Supplemental Figure 2). Proteomic and phosphoproteomic analyses of RapDTubuleanimals identified reduced expression or phosphorylation states of many amino acid transporters known to be highly expressed in the PT.37 Among these, SLC6A19 (B0AT1), SLC7A7 (y+LAT1), and SLC3A2 (4F2hc) had reduced expression levels, whereas diminished phosphor-ylation was found in SLC7A9 (b0,+AT) at position S15 and S18. B0AT1 mediates neutral amino acid transport from the lumi-nal compartment into the cellular lumen.37b0,+AT, a member of the heterodimeric amino acid transporter family, is the light subunit of SLC3A1 (rBAT), facilitating dibasic amino acid and cysteine transport into the cell. y+LAT1, another member of the heterodimeric amino acid transporter family, is exclusively expressed in the PT, whereas its companion 4F2hc is ubiqui-tously expressed. y+LAT1 and 4F2hc both assemble to form a heterodimeric amino acid transporter localizing to
the basolateral membrane of the PT predominantly in the S1 segment. Interestingly, this transporter mediates efflux of di-basic amino acids in exchange for neutral amino acids and sodium together with b0,+AT and its subunit rBAT. Loss of function mutations result in an inefficient urea cycle with consecutive hyperammonemia,37 which could, at least in part, explain increased urea values of RapRicDTubuleanimals. Phosphoproteomic analysis of RapDTubule renal cortexes revealed a strong reduction in the phosphorylation level of three phosphorylation sites on SLC4A4 (NBC1). Inactivating mutations of this gene were reported to lead to renal PT aci-dosis, another commonly observed feature of Fanconi syn-drome.6 Whether the identified phosphosites influence NBC1 transport needs additional determination on a molec-ular level.38,39In accordance with other reports using rapamy-cin, despite similar expression of the major PT phosphate transporters (NaPi-IIa, NaPi-IIc, and Pit-2), we detected phosphaturia in RapDTubule and RapRicDTubule animals.40,41 Influence of mTORC1 on so far unidentified interaction part-ners might be responsible for this enigmaticfinding.
In summary, mTORC1 deletion leads to a generalized PT dysfunction, mimicking the clinical diagnosis of Fanconi
syndrome (Figure 6). Our in-depth analysis reveals probable molecular pathways being affected in an organ-specific fash-ion. Additionally, we have identified a novel main kinase reg-ulator of PT function, opening new avenues for subsequent studies to unravel the kinase networks governing PT transport in health and disease.
CONCISE METHODS Animals and Treatments
All animal experiments were conducted according to the National Institutes of Health guide for the care and use of laboratory animals as well as the German law for the welfare of animals, and they were approved by local authorities (G-13/07, M-9/11, and M-4/13). Mice were housed in an SPF facility with free access to chow and water and a 12-hour day-night cycle. Breeding and genotyping were done according to standard procedures. Raptorfl/fl and Rictorfl/fl mice have been described previously42and were crossed to Pax8rtTA43
and TetOCre44animals. At 5 weeks of age, inducible animals and
respective controls (lacking either TetOCre or Pax8rtTA) received doxycycline hydrochloride (Fagron, Barsbuettel, Germany) via the Figure 6. Putative mTORC1–dependent regulation of proximal tubular transport and endocytosis. (A) mTORC1 seems to regulate apical and basolateral PT transport proteins. mTORC1 could directly phosphorylates SGLT2 and NBC1 to increase reabsorption of glucose and sodium and excretion of sodium and bicarbonate. mTORC1 might positively influence the expression level of the amino acid transporter B0AT1 and y+LAT-4F2hc and the phosphorylation status of b0,+AT1-rBAT to reabsorbfiltered amino acids and secrete
them into the bloodstream. (B) mTORC1 could regulate PT endocytosis, membrane biogenesis, vesicle trafficking, and cell polarity. mTORC1 likely controls apical membrane biogenesis and cell polarity by affecting expression level of DOCK8, apical clathrin–coated pit and vesicle formation, and endocytosis by affecting the expression levels of BCL-xL, SNX8, and RAB10 and phosphorylation level of
GBF-1. Also, it controls proper endoplasmic reticulum (ER) function and Golgi integrity again by influencing the expressions and phosphorylation levels of RAB10 and GBF-1, respectively.
drinking water (2 mg/ml with 5% sucrose protected from light) for a total of 14 days. To estimate recombination efficacy and identify cellular mosaicisms, we crossed the reporter mouse strain mT/mG with some Raptorfl/fl 3 Pax8rtTA 3 TetOCre mice (Supplemental Material).45
Presentation of Data and Statistical Analyses
Quantitative data are presented as means6SEM. Statistical compar-isons were performed using the GraphPad Prism Software Package 6 (GraphPad Software, La Jolla, CA). For statistical comparison, the Mann–Whitney U test, the two–tailed t test, and where appropriate, ANOVA were used. P values of,0.05 were considered statistically significant.
ACKNOWLEDGMENTS
We thank Temel Kilic, Barbara Joch, Benjamin Klormann, Anahita Rassi, Brigitte Scolari, Patricia Matthey, Danièla Grand-Habegger, Mélanie Bousquenaud, and Ruth Herzog for expert technical assis-tance. We also thank all members of our laboratories for helpful discussions and support.
This study was supported by German Research Foundation grants CRC 1140 (to F.G. and T.B.H.) and CRC 992 (to T.B.H.), the Hei-senberg Program (T.B.H.), European Research Council grant 616891 (to T.B.H.), BMBF–Joint Transnational grants 01KU1215 (to T.B.H.) and STOP-FSGS 01GM1518C (to T.B.H.), Excellence Initiative of the German Federal and State Governments EXC294, BIOSS II (T.B.H.), and the Swiss National Foundation (F.T.).
DISCLOSURES None.
REFERENCES
1. Smith HW: From Fish to Philosopher, Boston, Little, Brown, 1953 2. Zhuo JL, Li XC: Proximal nephron. Compr Physiol 3: 1079–1123, 2013 3. Makrides V, Camargo SM, Verrey F: Transport of amino acids in the
kidney. Compr Physiol 4: 367–403, 2014
4. Wagner CA, Rubio-Aliaga I, Biber J, Hernando N: Genetic diseases of renal phosphate handling. Nephrol Dial Transplant 29[Suppl 4]: iv45– iv54, 2014
5. Klootwijk ED, Reichold M, Helip-Wooley A, Tolaymat A, Broeker C, Robinette SL, Reinders J, Peindl D, Renner K, Eberhart K, Assmann N, Oefner PJ, Dettmer K, Sterner C, Schroeder J, Zorger N, Witzgall R, Reinhold SW, Stanescu HC, Bockenhauer D, Jaureguiberry G, Courtneidge H, Hall AM, Wijeyesekera AD, Holmes E, Nicholson JK, O’Brien K, Bernardini I, Krasnewich DM, Arcos-Burgos M, Izumi Y, Nonoguchi H, Jia Y, Reddy JK, Ilyas M, Unwin RJ, Gahl WA, Warth R, Kleta R: Mistargeting of peroxisomal EHHADH and inherited renal Fanconi’s syndrome. N Engl J Med 370: 129–138, 2014
6. Klootwijk ED, Reichold M, Unwin RJ, Kleta R, Warth R, Bockenhauer D: Renal Fanconi syndrome: Taking a proximal look at the nephron. Nephrol Dial Transplant 30: 1456–1460, 2015
7. Fanconi G: Der frühinfantile nephrotisch-glykosurische Zwergwuchs und hypophosphatämische Rachiti. Jahrbuch der Kinderheilkunde 147: 299, 1936
8. Christensen EI, Birn H, Storm T, Weyer K, Nielsen R: Endocytic recep-tors in the renal proximal tubule. Physiology (Bethesda) 27: 223–236, 2012
9. Christensen EI, Verroust PJ, Nielsen R: Receptor-mediated endocytosis in renal proximal tubule. Pflugers Arch 458: 1039–1048, 2009 10. Grahammer F, Wanner N, Huber TB: mTOR controls kidney epithelia in
health and disease. Nephrol Dial Transplant 29[Suppl 1]: i9–i18, 2014 11. Laplante M, Sabatini DM: mTOR signaling in growth control and
dis-ease. Cell 149: 274–293, 2012
12. Wullschleger S, Loewith R, Hall MN: TOR signaling in growth and metabolism. Cell 124: 471–484, 2006
13. Peterson TR, Sengupta SS, Harris TE, Carmack AE, Kang SA, Balderas E, Guertin DA, Madden KL, Carpenter AE, Finck BN, Sabatini DM: mTOR complex 1 regulates lipin 1 localization to control the SREBP pathway. Cell 146: 408–420, 2011
14. Sarbassov DD, Guertin DA, Ali SM, Sabatini DM: Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307: 1098– 1101, 2005
15. Ikenoue T, Inoki K, Yang Q, Zhou X, Guan KL: Essential function of TORC2 in PKC and Akt turn motif phosphorylation, maturation and signalling. EMBO J 27: 1919–1931, 2008
16. Rickheit G, Wartosch L, Schaffer S, Stobrawa SM, Novarino G, Weinert S, Jentsch TJ: Role of ClC-5 in renal endocytosis is unique among ClC exchangers and does not require PY-motif-dependent ubiquitylation. J Biol Chem 285: 17595–17603, 2010
17. Sorribas V, Markovich D, Hayes G, Stange G, Forgo J, Biber J, Murer H: Cloning of a Na/Pi cotransporter from opossum kidney cells. J Biol Chem 269: 6615–6621, 1994
18. Miller ML, Jensen LJ, Diella F, Jørgensen C, Tinti M, Li L, Hsiung M, Parker SA, Bordeaux J, Sicheritz-Ponten T, Olhovsky M, Pasculescu A, Alexander J, Knapp S, Blom N, Bork P, Li S, Cesareni G, Pawson T, Turk BE, Yaffe MB, Brunak S, Linding R: Linear motif atlas for phosphorylation-dependent signaling. Sci Signal 1: ra2, 2008
19. Golbaekdal K, Nielsen CB, Djurhuus JC, Pedersen EB: Effects of rapamycin on renal hemodynamics, water and sodium excretion, and plasma levels of angiotensin II, aldosterone, atrial natriuretic peptide, and vasopressin in pigs. Transplantation 58: 1153–1157, 1994 20. Morales JM, Andrés A, Dominguez-Gil B, Sierra MP, Arenas J, Delgado
M, Casal MC, Rodicio L: Tubular function in patients with hypokalemia induced by sirolimus after renal transplantation. Transplant Proc 35[Suppl]: 154S–156S, 2003
21. Morales JM, Wramner L, Kreis H, Durand D, Campistol JM, Andres A, Arenas J, Nègre E, Burke JT, Groth CG; Sirolimus European Renal Transplant Study Group: Sirolimus does not exhibit nephrotoxicity compared to cyclosporine in renal transplant recipients. Am J Trans-plant 2: 436–442, 2002
22. Ponticelli C, Graziani G: Proteinuria after kidney transplantation. Transpl Int 25: 909–917, 2012
23. Oroszlán M, Bieri M, Ligeti N, Farkas A, Meier B, Marti HP, Mohacsi P: Sirolimus and everolimus reduce albumin endocytosis in proximal tu-bule cells via an angiotensin II-dependent pathway. Transpl Immunol 23: 125–132, 2010
24. Gleixner EM, Canaud G, Hermle T, Guida MC, Kretz O, Helmstädter M, Huber TB, Eimer S, Terzi F, Simons M: V-ATPase/mTOR signaling regulates megalin-mediated apical endocytosis. Cell Reports 8: 10–19, 2014
25. Canaud G, Bienaimé F, Viau A, Treins C, Baron W, Nguyen C, Burtin M, Berissi S, Giannakakis K, Muda AO, Zschiedrich S, Huber TB, Friedlander G, Legendre C, Pontoglio M, Pende M, Terzi F: AKT2 is essential to maintain podocyte viability and function during chronic kidney disease. Nat Med 19: 1288–1296, 2013
26. Pohl M, Kaminski H, Castrop H, Bader M, Himmerkus N, Bleich M, Bachmann S, Theilig F: Intrarenal renin angiotensin system revisited: Role of megalin-dependent endocytosis along the proximal nephron. J Biol Chem 285: 41935–41946, 2010
27. Li H, Alavian KN, Lazrove E, Mehta N, Jones A, Zhang P, Licznerski P, Graham M, Uo T, Guo J, Rahner C, Duman RS, Morrison RS, Jonas EA: A Bcl-xL-Drp1 complex regulates synaptic vesicle membrane dynamics during endocytosis. Nat Cell Biol 15: 773–785, 2013
28. Ruusala A, Aspenström P: Isolation and characterisation of DOCK8, a member of the DOCK180-related regulators of cell morphology. FEBS Lett 572: 159–166, 2004
29. Harada Y, Tanaka Y, Terasawa M, Pieczyk M, Habiro K, Katakai T, Hanawa-Suetsugu K, Kukimoto-Niino M, Nishizaki T, Shirouzu M, Duan X, Uruno T, Nishikimi A, Sanematsu F, Yokoyama S, Stein JV, Kinashi T, Fukui Y: DOCK8 is a Cdc42 activator critical for interstitial dendritic cell migration during immune responses. Blood 119: 4451–4461, 2012 30. Zihni C, Munro PM, Elbediwy A, Keep NH, Terry SJ, Harris J, Balda MS,
Matter K: Dbl3 drives Cdc42 signaling at the apical margin to regulate junction position and apical differentiation. J Cell Biol 204: 111–127, 2014 31. D’Souza-Schorey C, Chavrier P: ARF proteins: Roles in membrane traffic
and beyond. Nat Rev Mol Cell Biol 7: 347–358, 2006
32. Dyve AB, Bergan J, Utskarpen A, Sandvig K: Sorting nexin 8 regulates endosome-to-Golgi transport. Biochem Biophys Res Commun 390: 109–114, 2009
33. Lv P, Sheng Y, Zhao Z, Zhao W, Gu L, Xu T, Song E: Targeted disruption of Rab10 causes early embryonic lethality. Protein Cell 6: 463–467, 2015
34. Shi A, Grant BD: Interactions between Rab and Arf GTPases regulate endosomal phosphatidylinositol-4,5-bisphosphate during endocytic recycling. Small GTPases 4: 106–109, 2013
35. Sabolic I, Vrhovac I, Eror DB, Gerasimova M, Rose M, Breljak D, Ljubojevic M, Brzica H, Sebastiani A, Thal SC, Sauvant C, Kipp H, Vallon V, Koepsell H: Expression of Na+-D-glucose cotransporter SGLT2 in rodents is kidney-specific and exhibits sex and species differences. Am J Physiol Cell Physiol 302: C1174–C1188, 2012
36. Ghezzi C, Wright EM: Regulation of the human Na+-dependent glu-cose cotransporter hSGLT2. Am J Physiol Cell Physiol 303: C348–C354, 2012
37. Camargo SM, Bockenhauer D, Kleta R: Aminoacidurias: Clinical and molecular aspects. Kidney Int 73: 918–925, 2008
38. May O, Yu H, Riederer B, Manns MP, Seidler U, Bachmann O: Short-term regulation of murine colonic NBCe1-B (electrogenic Na+/HCO3(-) cotransporter) membrane expression and activity by protein kinase C. PLoS One 9: e92275, 2014
39. Aeder SE, Martin PM, Soh JW, Hussaini IM: PKC-eta mediates glio-blastoma cell proliferation through the Akt and mTOR signaling path-ways. Oncogene 23: 9062–9069, 2004
40. Haller M, Amatschek S, Wilflingseder J, Kainz A, Bielesz B, Pavik I, Serra A, Mohebbi N, Biber J, Wagner CA, Oberbauer R: Sirolimus induced phosphaturia is not caused by inhibition of renal apical sodium phos-phate cotransporters. PLoS One 7: e39229, 2012
41. Kempe DS, Dërmaku-Sopjani M, Fröhlich H, Sopjani M, Umbach A, Puchchakayala G, Capasso A, Weiss F, Stübs M, Föller M, Lang F: Rapamycin-induced phosphaturia. Nephrol Dial Transplant 25: 2938– 2944, 2010
42. Bentzinger CF, Romanino K, Cloëtta D, Lin S, Mascarenhas JB, Oliveri F, Xia J, Casanova E, Costa CF, Brink M, Zorzato F, Hall MN, Rüegg MA: Skeletal muscle-specific ablation of raptor, but not of rictor, causes metabolic changes and results in muscle dystrophy. Cell Metab 8: 411– 424, 2008
43. Traykova-Brauch M, Schönig K, Greiner O, Miloud T, Jauch A, Bode M, Felsher DW, Glick AB, Kwiatkowski DJ, Bujard H, Horst J, von Knebel Doeberitz M, Niggli FK, Kriz W, Gröne HJ, Koesters R: An efficient and versatile system for acute and chronic modulation of renal tubular function in transgenic mice. Nat Med 14: 979–984, 2008
44. Eremina V, Jefferson JA, Kowalewska J, Hochster H, Haas M, Weisstuch J, Richardson C, Kopp JB, Kabir MG, Backx PH, Gerber HP, Ferrara N, Barisoni L, Alpers CE, Quaggin SE: VEGF inhibition and renal throm-botic microangiopathy. N Engl J Med 358: 1129–1136, 2008 45. Muzumdar MD, Tasic B, Miyamichi K, Li L, Luo L: A global
double-fluorescent Cre reporter mouse. Genesis 45: 593–605, 2007
